Protein expression and purification Mammalian cells

The RNA-guided CRISPR-Cas9 nuclease system has revolutionized the genome editing practices. For the most part, the Cas9-mediated genome editing is performed either via nonhomologous end joining (NHEJ) or homology-directed repair (HDR) in mammalian cells, However, designing of specific sgRNAs and minimizing off-target cleavage mediated mutagenesis are the major challenges in CRISPR-Cas based genome editing. To circumvent these issues, we can take advantages of many available tools and approaches for sgRNA construction and delivery.

The RNA-guided CRISPR-Cas9 nuclease system has revolutionized the genome editing practices. For the most part, the Cas9-mediated genome editing is performed either via nonhomologous end joining (NHEJ) or homology-directed repair (HDR) in mammalian cells, However, designing of specific sgRNAs and minimizing off-target cleavage mediated mutagenesis are the major challenges in CRISPR-Cas based genome editing. To circumvent these issues, we can take advantages of many available tools and approaches for sgRNA construction and delivery.

TUNEL assay is the cell death detection method where the biochemical marker of apoptosis is DNA fragmentation. The assay involves the microscopical detection of generated DNA fragments with free 3'-hydroxyl residues. in apoptotic cells using enzyme terminal deoxynucleotidyl transferase (TdT) which adds biotinylated nucleotides at the site of DNA breaks. Major challenges of this method involve proper access of the enzyme which could be hampered by poor permeabilization and/or excessive fixation with cross-linking fixative (common with archival tissue). This issue can be resolved by optimizing the incubation time with Proteinase K or CytoninTM.

Get tips on using EZViable™ Calcein AM Cell Viability Assay Kit (Fluorometric) to perform Live / Dead assay mammalian cells - rat brain microvascular endothelial cells

Get tips on using DNA-spin™ Plasmid DNA Purification Kit to perform Plasmid Isolation Enterobacteriaceae

Get tips on using LIVE/DEAD™ Fixable Blue Dead Cell Stain Kit, for UV excitation to perform Live / Dead assay mammalian cells - Spleen cells

Get tips on using Cell Counting Kit-8 to perform Live / Dead assay mammalian cells - rat nucleus pulposus

Get tips on using Attractene Transfection Reagent to perform DNA transfection Mammalian cells - Immortalized cell lines OVCAR-3

Get tips on using Live/Dead cell Staining Kit II to perform Live / Dead assay mammalian cells - H9C2